Cinnamate Salts Of A Beta-2 Adrenergic Agonist

ABSTRACT

This invention relates to cinnamate salts of 5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one.

The present invention relates to novel salts of a β₂ adrenergic agonist.In particular the invention relates to novel crystalline salts ofcompound (A) defined below. In addition the invention provides a processfor preparing said crystalline salts of compound (A) and topharmaceutical compositions containing them and their use in medicine.

β₂ Adrenergic receptor agonists are recognized as effective drugs forthe treatment of pulmonary diseases such as asthma and chronicobstructive pulmonary disease (including chronic bronchitis andemphysema). β₂ Adrenergic receptor agonists are also useful for treatingpremature labour, and are potentially useful for treating neurologicaldisorders and cardiac disorders.

U.S. Patent Application No. 60/535,784, and US 2005/0159448A1 (AttorneyDocket No. P-177-US1) describe compounds of formula:

wherein:

each of R¹, R², R³, and R⁴ is independently selected from the groupconsisting of hydrogen, hydroxy, amino, halo, —CH₂OH and —NHCHO, or R¹and R² taken together are selected from the group consisting of—NHC(═O)CH═CH—, —CH═CHC(═O)NH—, —NHC(═O)S; and —SC(═O)NH—;

one of R⁵ and R⁶ is —[X—C₁₋₆alkylenyl]_(n)—NR¹⁰R¹¹ orC₁₋₆alkylenyl-NR¹²R¹³, and the other of R⁵ and R⁶ is selected from thegroup consisting of hydrogen, hydroxy, C₁₋₄alkoxy, and C₁₋₄alkyl,wherein C₁₋₄alkyl is optionally substituted with halo, wherein

each X is independently selected from the group consisting of —O—, —NH—,—S—, —NHSO₂—, —SO₂NH—, —NHC(═O)—, and —C(═O)NH—;

each of R¹⁰, R¹¹, R¹², and R¹³ is independently hydrogen or C₁₋₄alkyl;or

R¹⁰ and R¹¹, together with the nitrogen atom to which they are attached,or

R¹⁰, together with the nitrogen atom to which it is attached and acarbon atom of the adjacent C₁₋₆alkylenyl, or R¹² and R¹³, together withthe nitrogen atom to which they are attached, or R¹², together with thenitrogen atom to which it is attached and a carbon atom of the adjacentC₁₋₆alkylenyl, form a heterocyclic or heteroaryl ring having from 5 to 7ring atoms, wherein the ring optionally contains an additionalheteroatom selected from oxygen, nitrogen, and sulfur, wherein nitrogenis optionally substituted with —S(O)₂—C₁₋₄alkyl; and

n is 1, 2, or 3; and

each of R⁷, R⁸, and R⁹ is independently hydrogen or C₁₋₆alkyl;

and pharmaceutically-acceptable salts, solvates and stereoisomersthereof.

Compounds of the aforementioned US applications are said to be potentand selective β₂ adrenergic receptor agonists.

Example 1 of the aforementioned US applications is the compound:

5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one(hereinafter referred to as Compound A), which may be depicted by thestructural formula (I):

We have now found certain novel salts of Compound A, viz. the cinnamate,4-methoxy cinnamate, 4-phenyl cinnamate and the 4-methyl cinnamate saltshereinafter known as the “salts of the invention”.

In a first aspect therefore the present invention provides a salt of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one,selected from a cinnamate, 4-methoxycinnamate, di-(4-phenylcinnamate)and 4-methyl cinnamate salt.

The invention also provides a salt of the compound of formula (I)hereinabove, wherein said salt is selected from a cinnamate,4-methoxycinnamate, di-(4-phenylcinnamate) and 4-methyl cinnamate salt.

In one embodiment the present invention provides a cinnamate salt of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one.

In another embodiment the present invention provides a 4-methoxycinnamate salt of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one.

In a further embodiment the present invention provides adi-(4-phenylcinnamate) salt of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one.

In a further embodiment the invention provides a 4-methyl cinnamate saltof5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one.

In a particular embodiment the invention provides a crystalline 4-methylcinnamate salt of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one.

In a particular embodiment the invention provides a crystallinecinnamate salt of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one.

In another embodiment the invention provides a crystalline 4-methoxycinnamate salt of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one.

In a particular embodiment the invention provides a crystallinedi-(4-phenylcinnamate) salt of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one.

We have found that salts of the invention may have varying hydrationstates. In a further embodiment the present invention encompasseshydrates of salts of the invention. Thus, for example the4-methylcinnamate salt may be obtained as a dihydrate, or as a hydrateassociated with 3.5 moles of water.

The present invention also encompasses solvates of salts of theinvention.

Salts of the invention may also exist in different stoichiometries. Thusfor example, the salt with 4-phenylcinnamic acid is formed with 2 molesof acid to each mole of Compound A.

It will be appreciated that the compound of formula (I), which isdepicted and named as the R-isomer with respect to the OH group, may bein admixture with the corresponding S-isomer and hence salts of theinvention may also exist as mixtures of said isomers. Thus for example,said admixture may contain at least 90%, for example at least 95% of theR-isomer.

The salts of the invention may be prepared by contacting a solution ofcompound A with an acid selected from cinnamic acid, 4-methoxy cinnamicacid, 4-phenylcinnamic acid or 4-methylcinnamic acid. Compound A may forexample be dissolved in an aqueous organic solution, for example in amixture of tetrahydrofuran and water, or an aqueous alcohol such asaqueous industrial methylated spirit. The reaction may be effected withstirring. The reaction temperature may be in the range from 0° C. to 50°C., for example from 15° C. to 30° C. Crystallisation may occurspontaneously or it may be induced, for example by scratching orseeding.

In one aspect of the present invention is provided a method forpreparing a 4-methoxy cinnamate salt of Compound A which comprisescontacting in solution Compound A and 4-methoxy cinnamic acid.

In another aspect the present invention provides a method for preparinga cinnamate salt of compound A which comprises contacting in solutionCompound A and cinnamic acid.

In a further aspect the present invention provides a method forpreparing a di-4-phenyl cinnamate salt of compound A which comprisescontacting in solution Compound A and 4-phenylcinnamic acid.

In a yet further aspect the present invention provides a method forpreparing a 4-methyl cinnamate salt of compound A which comprisescontacting in solution Compound A and 4-methyl cinnamic acid.

Compound A may itself be prepared using one of the general methodsdescribed in U.S. Patent Application No. 60/535,784, and US2005/0159448A1 as described hereinafter. Compound A may also be preparedby the specific method described by the Reference Example hereinafter.

The aforementioned US patent applications describes inter alia thefollowing general methods for the preparation of compounds definedtherein:

General Synthetic Procedures

Compounds of U.S. Patent Application No. 60/535,784, and US2005/0159448A1 can be prepared from readily available starting materialsusing the following general methods and procedures.

As will be apparent to those skilled in the art, conventional protectinggroups may be necessary to prevent certain functional groups fromundergoing undesired reactions. The choice of a suitable protectinggroup for a particular functional group, as well as suitable conditionsfor protection and deprotection, are well known in the art. For example,numerous protecting groups, and their introduction and removal, aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Third Edition, Wiley, New York, 1999, and references citedtherein.

In one method of synthesis, compounds are prepared as illustrated inScheme A. (The substituents and variables shown in the following schemeshave the definitions provided above unless otherwise indicated.)

where P¹ represents a hydroxy-protecting group, P² represents ahydroxy-protecting group, and L represents a leaving group, such asbromo.

As shown in Scheme A, a compound of formula 1 is first reacted with anaryl amine (2) to provide an intermediate of formula 3. Typically, thisreaction is conducted in an organic solvent in the presence of base anda transition metal catalyst and arylphosphine ligand with heating. Auseful catalyst for coupling of an aryl group to an aryl amine istris(dibenzylideneacetone)dipalladium(0) together withrac-2,2′-bis(diphenylphosphino)-1,1′-binapthyl. The reaction istypically heated at a temperature of between about 50° C. and about 120°C. for between about 0.25 and about 12 hours.

The protecting group P¹ is typically a silyl protecting group, which istypically removed from the intermediate of formula 3 using a fluoride oracid reagent, to provide an intermediate of formula 4. The protectinggroup P² is typically a benzyl protecting group, which is typicallyremoved from the intermediate of formula 4 by hydrogenation using apalladium on carbon catalyst, to provide the product.

An alternative method of preparing intermediate 3 is illustrated inScheme B.

The conditions for the coupling of intermediates 5 and 6 in Scheme B toproduce intermediate 3 are typically the same as those used to coupleintermediates 1 and 2 in Scheme A.

Yet another alternative method of preparing intermediate 3 isillustrated in Scheme C.

The reaction of Scheme C is typically conducted in a polar aproticsolvent in the presence of base. Typical suitable solvents includedimethylsulfoxide, dimethyl formamide, dimethylacetamide and the like.The reaction is typically heated at a temperature of between about 60°C. and about 140° C. for between about 0.25 and about 4 hours.

The compounds of formula 1 and 7 employed in the reactions described inthis application are readily prepared by procedures known in the art,and described, for example, in U.S. Pat. Nos. 6,653,323 B2 and 6,670,376B1, which are incorporated herein by reference, and references therein.Intermediate 5 can be prepared by reaction of intermediate 7 with2-(4-aminophenyl)ethylamine in an aprotic solvent with heating.

Intermediates 2 and 6 are available commercially or are prepared fromreadily available starting materials. For example, when R⁵ is—[O—C₁₋₆alkylenyl]_(n)—NR¹⁰R¹¹ and R⁶ is hydrogen, intermediate 2′, ofgeneral formula 2, can be prepared by the process of Scheme D

where R^(5a) is defined such that —OR^(5a) is—[O—C₁₋₆alkylenyl]_(n)—NR¹⁰R¹¹. As one example of suitable reactionconditions for Scheme D, the reaction is conducted in dimethylsulfoxidein the presence of sodium hydride. Alternatively, the reaction ofintermediate 9 with the compound HO—R^(5a) may be conducted in a solventsuch as tetrahydrofuran, in the presence of a base such as potassiumtert.-butoxide. Intermediate 10 may be converted into Intermediate 2′ byhydrogenation, for example using palladium on charcoal, in a solventsuch as industrial methylated spirits.

Further details regarding specific reaction conditions and otherprocedures for preparing Compound A are described in the Examples andReference Example below.

As indicated hereinabove compounds of U.S. Patent Application No.60/535,784, and US 2005/0159448A, including Compound A, are potent andselective β₂ adrenergic receptor agonists. The present inventionaccordingly also provides a method for the prophylaxis or treatment of aclinical condition in a mammal, such as a human, for which a selectiveβ₂-adrenoreceptor agonist is indicated, which comprises administrationof a therapeutically effective amount of a salt of the this invention.In particular, the present invention provides such a method for theprophylaxis or treatment of a disease associated with reversible airwaysobstruction such as asthma, chronic obstructive pulmonary disease(COPD), respiratory tract infection or upper respiratory tract disease.In a further aspect the present invention provides such a method for theprophylaxis or treatment of a clinical condition selected from prematurelabour, depression, congestive heart failure, skin diseases (e.g.inflammatory, allergic, psoriatic, and proliferative skin diseases),conditions where lowering peptic acidity is desirable (e.g. peptic andgastric ulceration) or muscle wasting disease.

In the alternative, there is also provided a salt of the invention foruse in medical therapy, particularly, for use in the prophylaxis ortreatment of a clinical condition in a mammal, such as a human, forwhich a selective β₂-adrenoreceptor agonist is indicated. In particular,there is provided a salt of the invention for the prophylaxis ortreatment of a disease associated with reversible airways obstructionsuch as asthma, chronic obstructive pulmonary disease (COPD),respiratory tract infection or upper respiratory tract disease. In afurther aspect, there is provided a salt of the invention for theprophylaxis or treatment of a clinical condition selected from prematurelabour, depression, congestive heart failure, skin diseases (e.g.inflammatory, allergic, psoriatic, and proliferative skin diseases),conditions where lowering peptic acidity is desirable (e.g. peptic andgastric ulceration) or muscle wasting disease.

The present invention also provides the use of a salt of the inventionin the manufacture of a medicament for the prophylaxis or treatment of aclinical condition for which a selective β₂-adrenoreceptor agonist isindicated, for example a disease associated with reversible airwaysobstruction such as asthma, chronic obstructive pulmonary disease(COPD), respiratory tract infection or upper respiratory tract disease.In a further aspect, there is provided a salt of the invention in themanufacture of a medicament for the prophylaxis or treatment of aclinical condition selected from premature labour, depression,congestive heart failure, skin diseases (e.g. inflammatory, allergic,psoriatic, and proliferative skin diseases), conditions where loweringpeptic acidity is desirable (e.g. peptic and gastric ulceration) andmuscle wasting disease.

The amount of a salt of the invention which is required to achieve atherapeutic effect will, of course, vary with the route ofadministration, the subject under treatment, and the particular disorderor disease being treated. Salts of the invention may be administered byinhalation at a dose of from 0.0005 mg to 10 mg, preferably from 0.005mg to 0.5 mg, for example, from 0.05 mg to 0.5 mg. The dose range foradult humans is generally from 0.0005 mg to 10 mg per day and preferablyfrom 0.01 mg to 1 mg per day, most preferrably from 0.05 mg to 0.5 mg.

While it is possible for a salt of the present invention, selected fromCompound A cinnamate, Compound A 4-methoxycinnamate, Compound Adi-(4-phenylcinnamate) and Compound A 4-methylcinnamate, to beadministered alone, it is preferable to present it as a pharmaceuticalformulation.

Accordingly, the present invention provides a pharmaceutical formulationcomprising Compound A cinnamate, and a pharmaceutically acceptablecarrier or excipient, and optionally one or more other therapeuticingredients.

The present invention provides a pharmaceutical formulation comprisingCompound A 4-methoxycinnamate, and a pharmaceutically acceptable carrieror excipient, and optionally one or more other therapeutic ingredients.

The present invention further provides a pharmaceutical formulationcomprising Compound A di-(4-phenyl cinnamate), and a pharmaceuticallyacceptable carrier or excipient, and optionally one or more othertherapeutic ingredients.

The present invention further provides a pharmaceutical formulationcomprising Compound A 4-methyl cinnamate, and a pharmaceuticallyacceptable carrier or excipient, and optionally one or more othertherapeutic ingredients.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous andintraarticular), inhalation (including fine particle dusts or mistswhich may be generated by means of various types of metered dosepressurised aerosols, nebulisers or insufflators), rectal and topical(including dermal, buccal, sublingual and intraocular) administrationalthough the most suitable route may depend upon for example thecondition and disorder of the recipient. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. All methods includethe step of bringing a salt of the invention (hereinafter also referredto as ‘active ingredient’) into association with the carrier whichconstitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

Dry powder compositions for topical delivery to the lung by inhalationmay, for example, be presented in capsules and cartridges of for examplegelatine, or blisters of for example laminated aluminium foil, for usein an inhaler or insufflator. Powder blend formulations generallycontain a powder mix for inhalation of the active ingredient and asuitable powder base (carrier/diluent/excipient substance) such asmono-, di or poly-saccharides (eg. lactose or starch). Use of lactose ispreferred. Dry powder compositions may also include, in addition to thedrug and carrier, a further excipient such as a sugar ester e.g.cellobiose octaacetate, calcium stearate or magnesium stearate.

Each capsule or cartridge may generally contain between 20 μg to 10 mgof a salt of the invention optionally in combination with anothertherapeutically active ingredient. Alternatively, the compound of theinvention may be presented without excipients. Packaging of theformulation may be suitable for unit dose or multi-dose delivery. In thecase of multi-dose delivery, the formulation can be pre-metered (eg asin Diskus, see GB 2242134, U.S. Pat. Nos. 6,632,666, 5,860,419,5,873,360 and 5,590,645 or Diskhaler, see GB 2178965, 2129691 and2169265, U.S. Pat. Nos. 4,778,054, 4,811,731, 5,035,237, the disclosuresof which are hereby incorporated by reference) or metered in use (eg asin Turbuhaler, see EP 69715 or in the devices described in U.S. Pat. No.6,321,747 the disclosures of which are hereby incorporated byreference). An example of a unit-dose device is Rotahaler (see GB2064336 and U.S. Pat. No. 4,353,656, the disclosures of which are herebyincorporated by reference). The Diskus inhalation device comprises anelongate strip formed from a base sheet having a plurality of recessesspaced along its length and a lid sheet hermetically but peelably sealedthereto to define a plurality of containers, each container havingtherein an inhalable formulation containing a salt of the inventionpreferably combined with lactose. Preferably, the strip is sufficientlyflexible to be wound into a roll. The lid sheet and base sheet willpreferably have leading end portions which are not sealed to one anotherand at least one of the said leading end portions is constructed to beattached to a winding means. Also, preferably the hermetic seal betweenthe base and lid sheets extends over their whole width. The lid sheetmay preferably be peeled from the base sheet in a longitudinal directionfrom a first end of the said base sheet. Alternatively, the formulationmay be presented if desired together with one or more other therapeuticagents in an inhalation device wherein the individual therapeutic agentsare administrable simultaneously but are stored separately (or wholly orpartly stored separately for triple combinations), e.g. in separatepharmaceutical compositions, for example as described in WO 03/061743.

Spray compositions for topical delivery to the lung by inhalation mayfor example be formulated as aqueous solutions or suspensions or asaerosols delivered from pressurised packs, such as a metered doseinhaler, with the use of a suitable liquefied propellant. Aerosolcompositions suitable for inhalation can be either a suspension or asolution and generally contain a salt of the invention optionally incombination with another therapeutically active ingredient and asuitable propellant such as a fluorocarbon or hydrogen-containingchlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes,e.g. dichlorodifluoromethane, trichlorofluoromethane,dichlorotetra-fluoroethane, especially 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. Carbon dioxideor other suitable gas may also be used as propellant. The aerosolcomposition may be excipient free or may optionally contain additionalformulation excipients well known in the art such as surfactants e.g.oleic acid or lecithin and cosolvents e.g. ethanol. Pressurisedformulations will generally be retained in a canister (e.g. an aluminiumcanister) closed with a valve (e.g. a metering valve) and fitted into anactuator provided with a mouthpiece.

Medicaments for administration by inhalation desirably have a controlledparticle size. The optimum particle size for inhalation into thebronchial system is usually 1-10 μm, preferably 2-5 μm. Particles havinga size above 20 μm are generally too large when inhaled to reach thesmall airways. To achieve these particle sizes the particles of theactive ingredient as produced may be size reduced by conventional meanse.g. by micronisation. The desired fraction may be separated out by airclassification or sieving. Preferably, the particles will becrystalline. When an excipient such as lactose is employed, generally,the particle size of the excipient will be much greater than the inhaledmedicament within the present invention. When the excipient is lactoseit will typically be present as milled lactose, wherein not more than85% of lactose particles will have a MMD of 60-90 μm and not more than15% will have a MMD of less than 15 μm.

Intranasal sprays may be formulated with aqueous or non-aqueous vehicleswith the addition of agents such as thickening agents, buffer salts oracid or alkali to adjust the pH, isotonicity adjusting agents oranti-oxidants.

Solutions for inhalation by nebulation may be formulated with an aqueousvehicle with the addition of agents such as acid or alkali, buffersalts, isotonicity adjusting agents or antimicrobials. They may besterilised by filtration or heating in an autoclave, or presented as anon-sterile product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The salts of the invention may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, lubricating, surface active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example saline or water-for-injection,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Formulations for rectal administration may be presented as a suppositorywith the usual carriers such as cocoa butter or polyethylene glycol.

Formulations for topical administration in the mouth, for examplebuccally or sublingually, include lozenges comprising the activeingredient in a flavoured basis such as sucrose and acacia ortragacanth, and pastilles comprising the active ingredient in a basissuch as gelatin and glycerin or sucrose an acacia.

Preferred unit dosage formulations are those containing an effectivedose, as hereinbefore recited, or an appropriate fraction thereof, ofthe active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

The compounds and pharmaceutical formulations according to the inventionmay be used in combination with or include one or more other therapeuticagents, for example selected from anti-inflammatory agents,anticholinergic agents (particularly an M₁, M₂, M₁/M₂ or M₃ receptorantagonist), other β₂-adrenoreceptor agonists, antiinfective agents(e.g. antibiotics, antivirals), or antihistamines. The invention thusprovides, in a further aspect, a combination comprising a salt of theinvention together with one or more other therapeutically active agents,for example selected from an anti-inflammatory agent (for example acorticosteroid or an NSAID), an anticholinergic agent, anotherβ₂-adrenoreceptor agonist, an antiinfective agent (e.g. an antibiotic oran antiviral), or an antihistamine. Examples of combinations are thosecomprising a salt of the invention together with a corticosteroid,and/or an anticholinergic, and/or a PDE-4 inhibitor. Suitablecombinations are those comprising one or two other therapeutic agents.

It will be clear to a person skilled in the art that, where appropriate,the other therapeutic ingredient(s) may be used in the form of salts,(e.g. as alkali metal or amine salts or as acid addition salts), orprodrugs, or as esters (e.g. lower alkyl esters), or as solvates (e.g.hydrates) to optimise the activity and/or stability and/or physicalcharacteristics (e.g. solubility) of the therapeutic ingredient. It willbe clear also that where appropriate, the therapeutic ingredients may beused in optically pure form.

Examples of anti-inflammatory agents include corticosteroids and NSAIDs.Corticosteroids which may be used in combination with the compounds ofthe invention are those oral and inhaled corticosteroids and theirpro-drugs which have anti-inflammatory activity. Examples include methylprednisolone, prednisolone, dexamethasone, fluticasone propionate,6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester,6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydro-furan-3S-yl) ester, beclomethasone esters (e.g.the 17-propionate ester or the 17,21-dipropionate ester), budesonide,flunisolide, mometasone esters (e.g. the furoate ester), triamcinoloneacetonide, rofleponide, ciclesonide, butixocort propionate, RPR-106541,and ST-126. Preferred corticosteroids include fluticasone propionate,6α,9α-difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester and6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester, more preferably6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester.

Examples of NSAIDs include sodium cromoglicate, nedocromil sodium,phosphodiesterase (PDE) inhibitors (e.g. theophylline, PDE4 inhibitorsor mixed PDE3/PDE4 inhibitors), leukotriene antagonists, inhibitors ofleukotriene synthesis, iNOS inhibitors, tryptase and elastaseinhibitors, beta-2 integrin antagonists and adenosine receptor agonistsor antagonists (e.g. adenosine 2a agonists), cytokine antagonists (e.g.chemokine antagonists) or inhibitors of cytokine synthesis.

Other β₂-adrenoreceptor agonists include salmeterol (e.g. as thexinofoate), salbutamol (e.g. as the sulphate or the free base),formoterol (e.g. as the fumarate), fenoterol or terbutaline and saltsthereof.

A salt of the invention may be used in combination with aphosphodiesterase 4 (PDE4) inhibitor or a mixed PDE3/PDE4 inhibitor. ThePDE4-specific inhibitor useful in this aspect of the invention may beany compound that is known to inhibit the PDE4 enzyme or which isdiscovered to act as a PDE4 inhibitor, and which are only PDE4inhibitors, not compounds which inhibit other members of the PDE familyas well as PDE4. Generally it is preferred to use a PDE4 inhibitor whichhas an IC₅₀ ratio of about 0.1 or greater as regards the IC₅₀ for thePDE4 catalytic form which binds rolipram with a high affinity divided bythe IC₅₀ for the form which binds rolipram with a low affinity. For thepurposes of this disclosure, the cAMP catalytic site which binds R and Srolipram with a low affinity is denominated the “low affinity” bindingsite (LPDE 4) and the other form of this catalytic site which bindsrolipram with a high affinity is denominated the “high affinity” bindingsite (HPDE 4). This term “HPDE4” should not be confused with the term“hPDE4” which is used to denote human PDE4.

A method for determining IC₅₀ ratios is set out in U.S. Pat. No.5,998,428 which is incorporated herein in full by reference as thoughset out herein. See also PCT application WO 00/51599 for anotherdescription of said assay.

Particular PDE4 inhibitors are those which have an IC₅₀ ratio of greaterthan 0.5, and particularly those compounds having a ratio of greaterthan 1.0. Examples of compounds include cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-oneandcis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];these are examples of compounds which bind preferentially to the lowaffinity binding site and which have an IC₅₀ ratio of 0.1 or greater.

Other Compounds of interest include:

Compounds set out in U.S. Pat. No. 5,552,438 issued 3 Sep., 1996; thispatent and the compounds it discloses are incorporated herein in full byreference. The compound of particular interest, which is disclosed inU.S. Pat. No. 5,552,438, iscis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid (also known as cilomalast) and its salts, esters, pro-drugs orphysical forms.

Other compounds of interest include AWD-12-281 from elbion (Hofgen, N.et al. 15th EFMC Int Symp Med Chem (September 6-10, Edinburgh) 1998,Abst P. 98; CAS reference No. 247584020-9); a 9-benzyladenine derivativenominated NCS-613 (INSERM); D-4418 from Chiroscience andSchering-Plough; a benzodiazepine PDE4 inhibitor identified as Cl-1018(PD-168787) and attributed to Pfizer; a benzodioxole derivativedisclosed by Kyowa Hakko in WO99/16766; K-34 from Kyowa Hakko; V-11294Afrom Napp (Landells, L. J. et al. Eur Resp J [Annu Cong Eur Resp Soc(September 19-23, Geneva) 1998] 1998, 12 (Suppl. 28): Abst P2393);roflumilast (CAS reference No 162401-32-3) and a phthalazinone(WO99/47505, the disclosure of which is hereby incorporated byreference) from Byk-Gulden; Pumafentrine,(−)-p-[(4aR*,10bS*)-9-ethoxy-1,2,3,4,4a,10b-hexahydro-8-methoxy-2-methylbenzo[c][1,6]naphthyridin-6-yl]-N,N-diisopropylbenzamidewhich is a mixed PDE3/PDE4 inhibitor which has been prepared andpublished on by Byk-Gulden, now Altana; arofylline under development byAlmirall-Prodesfarma; VM554/UM565 from Vernalis; or T-440 (TanabeSeiyaku; Fuji, K. et al. J Pharmacol Exp Ther, 1998, 284(1): 162), andT2585.

Other possible PDE-4 and mixed PDE3/PDE4 inhibitors include those listedin WO01/13953, the disclosure of which is hereby incorporated byreference.

Examples of anticholinergic agents are those compounds that act asantagonists at the muscarinic receptor, in particular those compoundswhich are antagonists of the M₁ and M₂ receptors. Exemplary compoundsinclude the alkaloids of the belladonna plants as illustrated by thelikes of atropine, scopolamine, homatropine, hyoscyamine; thesecompounds are normally administered as a salt, being tertiary amines.These drugs, particularly the salt forms, are readily available from anumber of commercial sources or can be made or prepared from literaturedata via, to wit:

Atropine—CAS-51-55-8 or CAS-51-48-1 (anhydrous form), atropinesulfate—CAS-5908-99-6; atropine oxide—CAS-4438-22-6 or its HClsalt—CAS-4574-60-1 and methylatropine nitrate—CAS-52-88-0.

Homatropine—CAS-87-00-3, hydrobromide salt—CAS-51-56-9, methylbromidesalt—CAS-80-49-9.

Hyoscyamine (d, l)—CAS-101-31-5, hydrobromide salt—CAS-306-03-6 andsulfate salt—CAS-6835-16-1.

Scopolamine—CAS-51-34-3, hydrobromide salt—CAS-6533-68-2, methylbromidesalt—CAS-155-41-9.

Particular anticholinergics include ipratropium (e.g. as the bromide),sold under the name Atrovent, oxitropium (e.g. as the bromide) andtiotropium (e.g. as the bromide) (CAS-139404-48-1). Also of interestare: methantheline (CAS-53-46-3), propantheline bromide (CAS-50-34-9),anisotropine methyl bromide or Valpin 50 (CAS-80-50-2), clidiniumbromide (Quarzan, CAS-3485-62-9), copyrrolate (Robinul), isopropamideiodide (CAS-71-81-8), mepenzolate bromide (U.S. Pat. No. 2,918,408),tridihexethyl chloride (Pathilone, CAS-4310-35-4), and hexocycliummethylsulfate (Tral, CAS-115-63-9). See also cyclopentolatehydrochloride (CAS-5870-29-1), tropicamide (CAS-1508-75-4),trihexyphenidyl hydrochloride (CAS-144-11-6), pirenzepine(CAS-29868-97-1), telenzepine (CAS-80880-90-9), AF-DX 116, ormethoctramine, and the compounds disclosed in WO01/04118, the disclosureof which is hereby incorporated by reference.

Examples of antihistamines (also referred to as H₁-receptor antagonists)include any one or more of the numerous antagonists known which inhibitH₁-receptors, and are safe for human use. All are reversible,competitive inhibitors of the interaction of histamine withH₁-receptors. The majority of these inhibitors, mostly first generationantagonists, have a core structure, which can be represented by thefollowing formula:

This generalized structure represents three types of antihistaminesgenerally available: ethanolamines, ethylenediamines, and alkylamines.In addition, other first generation antihistamines include those whichcan be characterized as based on piperizine and phenothiazines. Secondgeneration antagonists, which are non-sedating, have a similarstructure-activity relationship in that they retain the core ethylenegroup (the alkylamines) or mimic the tertiary amine group withpiperizine or piperidine. Exemplary antagonists are as follows:

Ethanolamines: carbinoxamine maleate, clemastine fumarate,diphenylhydramine hydrochloride, and dimenhydrinate.

Ethylenediamines: pyrilamine amleate, tripelennamine HCl, andtripelennamine citrate.

Alkylamines: chloropheniramine and its salts such as the maleate salt,and acrivastine.

Piperazines: hydroxyzine HCl, hydroxyzine pamoate, cyclizine HCl,cyclizine lactate, meclizine HCl, and cetirizine HCl.

Piperidines: Astemizole, levocabastine HCl, loratadine or itsdescarboethoxy analogue, and terfenadine and fexofenadine hydrochlorideor another pharmaceutically acceptable salt.

Azelastine hydrochloride is yet another H₁ receptor antagonist which maybe used in combination with a PDE4 inhibitor.

Examples of preferred anti-histamines include methapyrilene andloratadine.

The invention thus provides, in a further aspect, a combinationcomprising a salt of the invention together with a PDE4 inhibitor.

The invention thus provides, in a further aspect, a combinationcomprising a salt of the invention together with a corticosteroid, e.g.fluticasone propionate,6α,9α-difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester.

The invention provides, in a further aspect, a combination comprising asalt of the invention together with an anticholinergic, e.g.ipratropium, oxitropium or tiotropium.

The invention provides, in a further aspect, a combination comprising asalt of the invention together with an antihistamine.

The invention provides, in a further aspect, a combination comprising asalt of the invention together with a PDE4 inhibitor and acorticosteroid, e.g. with an antihistamine and a corticosteroid asdescribed hereinabove.

The invention provides, in a further aspect, a combination comprising asalt of the invention together with an anticholinergic and a PDE-4inhibitor, e.g. with a PDE4 inhibitor and an anticholinergic asdescribed hereinabove.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above together with aphysiologically acceptable diluent or carrier represent a further aspectof the invention.

The individual compounds of such combinations may be administered eithersequentially or simultaneously in separate or combined pharmaceuticalformulations. Appropriate doses of known therapeutic agents will bereadily appreciated by those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an x-ray powder diffraction pattern of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onecinnamate.

FIG. 2 shows an x-ray powder diffraction pattern of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one4-methoxycinnamate.

FIG. 3 shows an x-ray powder diffraction pattern of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onedi-(4-phenylcinnamate).

FIG. 4 shows an x-ray powder diffraction pattern of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one4-methylcinnamate (prepared according to the method of Example 4, Method1)

FIG. 5 shows an x-ray powder diffraction pattern of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one4-methylcinnamate (prepared according to the method of Example 4, Method2).

For a better understanding of the invention, the following Examples aregiven by way of illustration.

Abbreviations

DMSO Dimethylsulfoxide

DSC Differential scanning calorimetry

H₂O Water

HPLC High pressure liquid chromatography

TGA Thermogravimetric analysis

THF Tetrahydrofuran

TLC Thin layer chromatography

TMS Trimethylsilane

XRPD X-ray powder diffraction

IMS Industrial methylated spirits

The following analytical methods were used in respect of Examples 1-6:

XRPD analysis was performed on a Panalytical X-ray powderdiffractometer, model X'Pert Pro PW3040/60, serial number DY1850. Themethod runs from 2 to 40 degrees 2-Theta with a 0.0167 degree 2-Thetastep size and a 31.75 second collection time at each step using anX'celerator detector.

1H NMR spectra were acquired on a 400 MHz Bruker DPX400 spectrometer.Sample was dissolved in dmso-d6 and chemical shifts were reported in ppmrelative to the TMS signal at 0 ppm.

Where necessary reaction mixtures were purified using Biotage packedsilica columns.

For Example 1 the differential scanning calorimetry analysis wasobtained using a Perkin Elmer Pyris 1, serial number 537N9062304.Samples were weighed into an aluminium pan, an aluminium lid placed ontop of the sample and compressed with a brass rod. An empty pan and lidserved as reference. Samples were equilibrated at 30° C. and heated at10° C./min to 300° C. The instrument was calibrated using indium, tinand lead standards.

For Examples 2, 3 and 4, the differential scanning calorimetry analysiswas obtained using a TA Instruments 2920 MDSC, serial number M2920-234.Samples were weighed into an aluminium pan, a vented aluminium lidplaced on top of the sample and compressed with a brass rod. An emptypan and lid served as reference. Samples were equilibrated at 30° C. andheated at 10° C./min to a temperature between 250 and 350° C. Theinstrument was calibrated using indium and lead standards.

For Example 1, the thermogravimetric analysis was obtained using aPerkin Elmer Pyris 1, serial number 537N9031106. Samples were placedinto a tared aluminium pan and then positioned on a platinum crucible.Samples were heated from 30° C. at 10° C./min to 300° C. The instrumentwas calibrated for temperature using the Curie point of nickel andalumel.

For Examples 2, 3 and 4, the thermogravimetric analysis was obtainedusing a TA Instruments 2950 TGA, serial number HA2950-226. Samples wereplaced into a tared aluminium pan and then positioned on a platinumcrucible. Samples were heated from ambient at 10° C./min to atemperature between 250 and 350° C. The instrument was calibrated fortemperature using the Curie point of nickel and alumel.

REFERENCE EXAMPLE

Unless noted otherwise, in this reference example reagents, startingmaterial and solvents were purchased from commercial suppliers, forexample Sigma-Aldrich (St. Louis, Mo.), J. T. Baker (Phillipsburg,N.J.), and Honeywell Burdick and Jackson (Muskegon, Mi), and usedwithout further purification; reactions were run under nitrogenatmosphere; reaction mixtures were monitored by thin layerchromatography (silica TLC), analytical high performance liquidchromatography (anal. HPLC), or mass spectrometry; reaction mixtureswere commonly purified by flash column chromatography on silica gel, orby preparative HPLC using the general protocol described below; NMRsamples were dissolved in deuterated solvent (CD₃OD, CDCl₃, or DMSO-d6),and spectra were acquired with a Varian Gemini 2000 instrument (300 MHz)under standard parameters; and mass spectrometric identification wasperformed by an electrospray ionization method (ESMS) with a PerkinElmer instrument (PE SCIEX API 150 EX).

Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one

a. Preparation of 4-(2-amino-2-methyl-Propoxy)-phenylamine hydrochloride

A vigorously stirred slurry of sodium hydride (60% dispersion in mineraloil, 11.32 g, 0.28 mol) in dimethylsulfoxide (400 mL) was heated at 45°C. for 1 h. To this slurry was then added neat2-amino-2-methyl-1-propanol (25.3 g, 1 equiv). The reaction mixture waswarmed to 75° C. for 1 h then cooled to 20° C. in an ice bath.1-Fluoro-4-nitrobenzene (40 g, 1 equiv) was added slowly, maintainingthe temperature below 30° C., and the resulting dark red solution wasstirred at room temperature for a further 1 h. The reaction was quenchedwith water (1000 mL), extracted with dichloromethane (500 mL), and theorganic layer washed (1:1 saturated aqueous sodium chloride:water, 1000mL). The product was precipitated by addition of 3M hydrochloric acid(400 mL) to the organic layer. The resulting orange solid was thenfiltered and washed with dichloromethane until the filtrate wascolourless.

The solid material was immediately transferred to a hydrogenation flask.

Palladium (10% w/w on carbon, 50% w/w water) was added, followed bymethanol (500 mL). The slurry was shaken vigorously under 3 atmospheresof hydrogen gas for 16 h. The catalyst was then filtered, the solventremoved under reduced pressure, and the resulting solid dried byazeotroping with toluene (3×150 mL) to afford the title intermediate asa white solid (40 g, 0.18 mol, 65%).

b. Preparation of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-8-benzyloxy-1H-quinolin-2-one

A mixture of the product of step a (23.2 g, 1.1 equiv),8-benzyloxy-5-{(R)-2-[2-(4-bromo-phenyl)-ethylamino-1-(tert-butyl-dimethyl-silanyloxy)-ethyl}-1H-quinolin-2-onehydrochloride (66.0 g, 0.1 mol), and sodium tert-butoxide (54.0 g, 5.5equiv) in toluene (600 mL) was stirred at 90° C. until a homogenoussolution was obtained. Palladium tris(dibenzylideneacetone) (1.4 g,0.015 equiv) was added, followed byrac-2,2′-bis(diphenylphosphino)-1,1′-binapthyl (2.87 g, 0.045 equiv).The reaction mixture was stirred at 90° C. for 3 h, then allowed tocool. The solution was washed with water (100 mL), 1:1 saturated aqueoussodium chloride:water (100 mL), then dried over sodium sulfate. Thesolvent was removed under reduced pressure to afford the titleintermediate as a dark brown solid (40 g crude), which was used withoutfurther purification.

c. Preparation of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-benzyloxy-1H-quinolin-2-one

The product of the previous step was treated with triethylaminetrihydrofluoride (36 g) in 2-propanol (500 mL)/ethanol (100 mL) at roomtemperature for 16 h. The mixture was concentrated under reducedpressure to one third of its original volume. 1M aqueous sodiumhydroxide (500 mL) was added, followed by acetonitrile (500 mL) andisopropyl acetate (500 mL). The aqueous layer was removed and theorganic phase washed with 1:1 saturated aqueous sodium chloride:water(400 mL) then saturated aqueous sodium chloride (400 mL). The organicswere dried over sodium sulfate and the solvent removed in vacuo toafford the title intermediate (50 g crude) as a brown solid, which wasused without further purification.

d. Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one

Palladium hydroxide (10 g, 20% w/w on carbon, 50% w/w water) was addedto the product from the previous reaction, followed by ethanol (500 mL).The slurry was stirred vigorously under an atmosphere of hydrogen gasfor 8 h. The catalyst was filtered and the filtrate concentrated underreduced pressure to afford the title compound (40 g), which was purifiedby reverse phase HPLC and isolated as its trifluoroacetate salt bylyophilization.

1H NMR (300 MHz, DMSO-d6)

δ (ppm): 10.4 (s, 1H), 9.3 (br s, 1H), 8.7 (br s, 1H), 8.15 (m, 2H), 7.8(br s, 1H), 7.03 (d, 1H, J=8.2), 6.76-7.01 (m, 10H), 6.42 (d, 1H,J=9.6), 6.1 (brs, 1H), 5.33 (d, 1H, J=9.1), 3.8 (s, 2H), 2.7-3.1 (m,6H), 1.21 (s, 6H); m/z: [M+H+] calculated for C29H34N4O4, 503.3; found503.5.

Example 15-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onecinnamate (Unquantified Hydrate)

5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one(0.11 g) was slurried in aqueous tetrahydrofuran (6:4 THF:H₂O vol/vol,1.0 mL). Trans-cinnamic acid (0.03 g) in aqueous THF (6:4 THF:H₂Ovol/vol, 0.1 mL) was made up, most of the acid dissolved. A portion ofthe acid mixture was added to the free base solution which was seededwith a slurry of the salt in aqueous THF (6:4 THF:H₂O vol/vol). The seedsolution had been made by a similar method to this crystallisation.Since a thick slurry did not ensue, the remaining trans cinnamic acidmixture and more seed slurry was added.

Having stirred at room temperature overnight, the slurry was filtered.The filtered solid was washed with aqueous THF (2:1 THF:H₂O vol/vol,2×0.2 mL) and dried in vacuo at 45° C. overnight to give the titlecompound.

Yield (not accounting for water content) 63% th

400 MHz NMR in d6-DMSO. TMS as reference at 0 ppm.

δ (ppm): 1.20 (6H) s; 2.62 (2H) t J=7.3 Hz; 2.70-2.85 (4H) m; 3.72 (2H)s; 5.07 (1H) m; 6.50 (1H) d J=15.9 Hz; 6.50 (1H) d J=9.8 Hz; 6.85 (2H) dJ=8.6 Hz; 6.87 (2H) d J=9.1 Hz; 6.94 (1H) d J=8.1 Hz; 6.99 (2H) d J=8.8Hz; 7.00 (2H) d J=8.3 Hz; 7.06 (1H) d J=8.1 Hz; 7.31-7.42 (4H) m;7.57-7.61 (2H) m; 7.76 (1H) s; 8.18 (1H) d J=10.0 Hz

DSC: The sample exhibits an endotherm with an onset of around 60° C.This is followed by a second endotherm with an onset of 106° C. and asubsequent decomposition.

TGA: The sample exhibits a weight loss of 2.8% w/w from ambient toapproximately 63° C. This is followed by a second weight loss of 5.4%w/w from 63° C. to approximately 108° C. Subsequent weight loss is dueto decomposition.

XRPD analysis of the product is shown in FIG. 1

Example 25-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one4-methoxy cinnamate (Unquantified Hydrate)

5-[(R)-2-(2-{4-[(4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one(0.101 g) was slurried in aqueous tetrahydrofuran (1:1 THF:H₂O vol/vol,1.2 mL). 4-Methoxycinnamic acid (0.036 g, predominantly trans) was addedto the mixture. A solution formed before crystallisation spontaneouslyoccurred. The resultant slurry was stirred overnight at room temperatureand then filtered. The filtered solid was washed with aqueoustetrahydrofuran (1:1 THF:H₂O vol/vol, 3×0.2 mL) and dried at 45° C. invacuo overnight to give the title compound.

Yield (not accounting for water content) 69% th, 96% w/w

400 MHz NMR in d6-DMSO. TMS as reference at 0 ppm.

δ (ppm): 1.17 (6H) s; 2.61 (2H) t J=7.09 Hz; 2.69-2.83 (4H) m; 3.69 (2H)s; 3.78 (3H) s; 5.05 (1H) m; 6.36 (1H) d J=15.9 Hz; 6.49 (1H) d J=10.0Hz; 6.85 (2H) d J=8.1 Hz; 6.87 (2H) d J=7.8 Hz; 6.91-7.02 (7H) m; 7.06(1H) d J=8.1 Hz; 7.41 (1H) d J=15.9 Hz; 7.56 (2H) d J=8.6 Hz; 7.72 (1H)s; 8.18 (1H) d J=10.0 Hz.

DSC: The sample shows an endothermic event with an onset of around 112°C. followed by decomposition.

TGA: The sample exhibits a weight loss of 5.7% w/w from ambient toapproximately 125° C. This is followed by a second weight loss of 2.3%w/w from 125° C. to approximately 170° C. due to decomposition.

XRPD analysis of the product is shown in FIG. 2

Example 35-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-oneDi-(4-phenylcinnamate) (UnquantifiedH)

5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one(0.108 g) was slurried in aqueous tetrahydrofuran (1:1 THF:H₂O, 1.2 mL).4-Phenylcinnamic acid (0.049 g) was added to the stirred mixture at roomtemperature and spontaneous crystallisation occurred. The mixture wasstirred for over 1 hour and then filtered. The filtered solid was washedwith aqueous tetrahydrofuran (1:1 THF:H₂O, 2×0.2 mL). Having been leftto sit at ambient conditions for several hours, the wet solid was driedovernight at 60° C. in vacuo to give the title compound.

Yield (based on free base input, not accounting for water content)=33%th, 62% w/w

Yield (based on acid, not accounting for water content)=66% th

400 MHz NMR in d6-DMSO. TMS as reference at 0 ppm.

δ (ppm): 1.23 (6H) s; 2.65 (2H) t J=7.3 Hz; 2.75-2.88 (4H) m; 3.76 (2H)s; 5.10 (1H) m; 6.50 (1H) d J=9.8 Hz; 6.56 (2H) d J=15.9 Hz; 6.86 (2H) dJ=8.6 Hz; 6.88 (2H) d J=9.1 Hz; 6.96 (1H) d J=8.1 Hz; 6.99 (2H) d J=8.8Hz; 7.00 (2H) d J=8.8 Hz; 7.07 (1H) d J=8.1 Hz; 7.38 (2H) t J=7.3 Hz;7.45-7.49 (4H) m; 7.51 (2H) d J=10.3 Hz; 7.67-7.77 (13H) m; 8.19 (1H) dJ=10.0 Hz.

DSC: The sample shows an endothermic event with an onset of around 60°C. This is followed by a second endotherm with an onset of 145° C. andsubsequent decomposition.

XRPD analysis of the product is shown in FIG. 3

Example 45-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one4-methyl cinnamate

Method 1

5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one(0.21 g) was dissolved in a mixture of tetrahydrofuran (1.2 mL) andwater (1.2 mL). 4-methyl cinnamic acid (0.07 g, predominantly trans) wasadded to the stirred solution at room temperature and it dissolved.After approximately 10 minutes, crystallisation occurred. The slurry wasstirred overnight and filtered. The cake was washed with aqueousTetrahydrofuran (1:1 THF:water, 2×0.4 mL, 1×0.2 mL) to afford the titlecompound which was dried overnight under vacuum at 45° C.

400 MHz NMR in D6-DMSO. D5-DMSO as reference at 2.5 ppm.

δ (ppm): 1.22 (6H) s; 2.30 (3H) s; 2.64 (2H) t J=6.6 Hz; 2.72-2.86 (4H)m; 3.74 (2H) s; 5.09 (1H) m; 6.43(1H) d J=15.9 Hz; 6.49 (1H) d J=10.0Hz; 6.86 (4H) m; 6.93-7.03 (5H) m; 7.06 (1H) d J=7.8 Hz; 7.17 (2H) dJ=7.8 Hz; 7.34 (1H) d J=15.9 Hz; 7.45 (2H) d J=7.8 Hz; 7.75 (1H) s; 8.19(1H) d J=10.0 Hz

Expected yield 75% th, 100% w/w

DSC: The sample shows an endothermic event with an onset of around 106°C. This is followed by decomposition.

TGA: The sample exhibits a weight loss of 6.1% w/w from ambient toapproximately 120° C. This is followed by a second weight loss of 2.0%w/w and decomposition.

The XRPD pattern of this product is shown in FIG. 4.

Method 2

5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one(17.77 g) was dissolved in a mixture of tetrahydrofuran (89 mL) andwater (89 mL). 4-methyl cinnamic acid (6.02 g, predominantly trans) wasweighed out and about ¼ was added to the free base solution followed bysome seed crystals. The mixture was stirred and the remaining acid wasadded in portions over the following 1½ hours. After a further 4 hoursstirring, the slurry was filtered. The cake was washed with aqueoustetrahydrofuran (1:1 THF:water, 36 mL) and then tetrahydrofuran (2×18mL) to afford the title compound which was dried overnight at 40-50° C.under vacuum.

400 Mhz NMR in CD₃OD. TMS as 0 ppm reference.

δ (ppm): 1.39 (6H) s; 2.32 (3H) s; 2.79 (2H) t J=7.2 Hz; 2.92-3.03 (4H)m; 3.87 (2H) s; 5.25 (1H) d of d J=3.9 and 8.8 Hz; 6.44 (1H) d J=15.9Hz; 6.63(1H) d J=9.8 Hz; 6.86-6.93 (4H) m; 6.96 (1H) d J=8.3 Hz;6.99-7.05 (4H) m; 7.15 (2H) d J=7.8 Hz; 7.19 (1H) d J=8.3 Hz; 7.36 (1H)d J=15.9 Hz; 7.38 (2H) d J=7.6 Hz; 8.34 (1H) d J=9.8 Hz

Expected yield: 67% th, 95% w/w

DSC: The sample shows an endothermic event with an onset of around 67°C. This is followed by second endotherm with an onset of around 113° C.and then decomposition.

TGA: The sample exhibits a weight loss of 3.7% w/w from ambient toapproximately 75° C. This is followed by a second weight loss of 5.1%w/w from 75 to approximately 125° C., a third weight loss of 2.1% w/wand decomposition.

The XRPD pattern of this product is shown in FIG. 5.

Example 5 (a) Formation of 3.5 hydrate of 4-methyl cinnamate salt

4-Methyl cinnamic acid (33.7 g, 0.85 eq) was added to a solution of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onefree base (which may be prepared according to the method of Ex 6(d)) inaqueous IMS. The mixture was seeded but the seed dissolved. Water (3×50mL, 3×0.34 vols) was added until seeding gave a slow crystallisation.After 1.5 hours additional water (50 mL, 0.34 vols) was added and themixture stirred at room temperature overnight. More water (50 ml, 0.34vols) was added to the mixture. After 4 hours the slurry was filtered.The cake was washed with 15% vol/vol aqueous IMS (3×150 mL, 3×1 vol),taking the first wash to ‘dry land’ (no visible liquid on cake) butdeliquoring the cake significantly after the remaining two washes. Thecake was dried in a vacuum oven at ˜100 mbar at 43° C. for approximately24 hours to afford the title compound (127 g), Yield 71% th, 88% w/w TheXRPD of the product matches that shown previously in FIG. 5.

(b) 4-Methyl cinnamate dihydrate

4-methyl cinnamate 3.5 hydrate (134.9 g), prepared by the above method,was further dried in a vacuum oven at 40° C. with a vacuum pump toreduce to significantly below 100 mbar. Most of the resulting solid(67.7 g) was slurried in 5% vol/vol aqueous IMS (500 mL, 7.4 vols) for 7hours. The slurry was filtered and the cake washed with 5% vol/volaqueous IMS (70 mL×2, 2×1 vol). The cake was dried in a vacuum oven (42°C., ˜100 mbar) overnight to afford the title compound (61.8 g).

Yield from 3.5 hydrate 90% th, 86% w/w

XRPD of the product matches that shown previously in FIG. 4.

Example 6 Alternative Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one

a. {1,1-dimethyl-2-[(4-nitrophenyl)oxy]ethyl}amine

A solution of 2-amino-2-methyl-1-propanol (488 g, 5.47 mol) intetrahydrofuran (1.46 L) was added over 35 mins to a solution ofpotassium tert-butoxide (634 g, 5.65 mol) in tetrahydrofuran (2.95 L) at2±3° C. The mixture was maintained at this temperature for a further 55minutes before 4-fluoronitrobenzene (551 mL, 3.90 mol) was added over 1hour 25 mins keeping the temperature below 10° C. The mixture was warmedto 20±3° C. and stirred for 2 hours. Water (1.95 L) and Isopropylacetate (1.95 L) were charged to the mixture which was stirred, settledand separated. The organic phase was washed again with water (1.95 L)before 5M hydrochloric acid (1.95 L) was charged, keeping thetemperature below 25° C. Isooctane (1.95 L) was added and the layersseparated. 5M hydrochloric acid (322 mL) was added to the aqueous phasewhich was then reduced to approximately 3.9 L by vacuum distillation,causing spontaneous crystallisation, and stirred at room temperatureovernight. Filtration, washing with water (2×488 mL) and drying in vacuoat around 48° C. afforded the title intermediate as a white solid (972.1g, 72% th).

b. {4-[(2-amino-2-methylpropyl)oxy]phenyl}amine dihydrochloride

{1,1-dimethyl-2-[(4-nitrophenyl)oxy]ethyl}amine (752.5 g, 3.05 mol), 5%Pd/C (Escat 160, 30.1 g, 53.6% w/w water, 6.6 mmol Pd) and IMS (6.0 L)were stirred under hydrogen at atmospheric pressure until the exothermceased. The catalyst was filtered off through a CUNO celite filter whichwas washed with IMS (3.0 L). Concentrated hydrochloric acid (300 mL) wasadded to the combined organic solution over 45 minutes giving rise to aslurry which was stirred at room temperature overnight. Filtration,washing of the cake with IMS (2×1.12 L) and drying in vacuo at 37° C.afforded the title intermediate as a pink solid (646 g, 84% th).

c5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-8-benzyloxy-1H-quinolin-2-one

1,4-Dioxane (4.33 L) was added to a mixture of{4-[(2-amino-2-methylpropyl)oxy]phenyl}amine dihydrochloride (406.7 g,1.61 mol),8-benzyloxy-5-{(R)-2-[2-(4-bromo-phenyl)-ethylamino-1-(tert-butyl-dimethyl-silanyloxy)-ethyl}-1H-quinolin-2-onehydrochloride (865.4 g, 1.34 mol), sodium tert-butoxide (900 g, 9.36mol), bis(dibenzylideneacetone) palladium (9.4 g) and BINAP(rac-2,2′-bis(diphenylphosphino)-1,1′-binapthyl, 15.58 g). The mixturewas stirred and heated at 87±3° C. for 4 hours. The mixture was cooledto room temperature, quenched with water (2.60 L) and passed through aCUNO zetacarbon filter. Methyl acetate (2.60 L) and saturated sodiumchloride solution (made with 1.66 L water) were added, the solutionmixed and the phases settled and separated. The organic phase was washedwith sodium chloride solution (623 g in 2.84 L water). Methanol (3.46 L)and concentrated hydrochloric acid (735.6 mL) were added to the reactionmixture which was heated to 50±3° C. for 17 hours before returning to20±3° C. 5M sodium hydroxide solution (3.46 L) methyl acetate (3.46 L)and saturated brine (made with 1.1 L water) were added, the mixturestirred and the layers separated. The organic phase was washed withsodium chloride solution (622.8 g in 2.84 L water) and evaporated to adark foam on a rotary evaporator (851.2 g, crude yield 107% th, 98%w/w).

The foam was redissolved in methanol (1.2 L). A portion of the solution(1069 g) that contained 500 g of the crude material was injected onto acolumn in a biotage flash 150 system and eluted isocratically (99 partsmethanol, 1 part 2M ammonia in methanol). The product containingfractions were grouped by purity, clean fractions and those with lowlevel impurities were combined and evaporated to give the title compoundas 2 yellow foams (combined mass 305.6 g, 0.51 mol, yield from8-benzyloxy-5-{(R)-2-[2-(4-bromo-phenyl)-ethylamino-1-(tert-butyl-dimethyl-silanyloxy)-ethyl}-1H-quinolin-2-onehydrochloride, 65% th)

d. Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one

5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-8-benzyloxy-1H-quinolin-2-one(145 g 0.24 mol), made and purified in a similar way to that describedin section c above, palladium hydroxide on carbon (29 g 20% w/w Pd ondry basis, wet) and IMS (1.075 L) were stirred under hydrogen atatmospheric pressure and heated with a water bath at 45° C. for a totalof 11.5 hours (over 2 days). The catalyst was filtered off on a bed ofcelite which wastwice with IMS (300 mL then 150 mL) and then withaqueous IMS (2:1 IMS:water, 150 mL). This aqueous alcoholic free basesolution could then be used directly for salt formation or dried andevaporated to give a solid which could be re-dissolved for saltformation.

1. A salt of the compound of formula (I):

wherein said salt is selected from a cinnamate, 4-methoxycinnamate,di-(4-phenylcinnamate) and 4-methyl cinnamate salt. 2.5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one4-methyl cinammate. 3.5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onecinnamate. 4.5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one4-methoxy cinnamate. 5.5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onedi-(4-phenyl cinnamate).
 6. A 4-methyl cinnamate salt of the compound offormula:


7. A cinnamate salt of the compound of formula:


8. A 4-methoxy cinnamate salt of the compound of formula:


9. A 4-phenyl cinnamate of the compound of formula:


10. Crystalline5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one4-methyl cinammate.
 11. Crystalline5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onecinammate.
 12. Crystalline5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one4-methoxy cinnamate.
 13. Crystalline5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-oneDi-4-phenyl cinnamate.
 14. Crystalline 4-methyl cinnamate salt of thecompound of formula:


15. Crystalline cinnamate salt of the compound of formula:


16. Crystalline 4-methoxy cinnamate salt of the compound of formula:


17. Crystalline 4-phenyl cinnamate salt of the compound of formula:


18. A pharmaceutical formulation comprising a compound according toclaim 1 and a pharmaceutically acceptable carrier or excipient, andoptionally one or more other therapeutic ingredients.
 19. A combinationcomprising a compound according to claim 1 and one or more othertherapeutic ingredients.
 20. (canceled)
 21. A method for the prophylaxisor treatment of a clinical condition in a mammal, such as a human, forwhich a selective β₂-adrenoreceptor agonist is indicated, whichcomprises administration of a therapeutically effective amount of acompound according to claim
 1. 22. (canceled)
 23. A method for preparinga crystalline compound according to claim 1 which method comprisescontacting in solution5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onewith an acid selected from cinnamic acid, 4-methoxycinnamic acid,di-(4-phenylcinammic acid) and 4-methylcinnamic acid.